Microfluidic Generation Of Multicellular Tumor Spheroids Mimicking Microenvironment Of Tumor

Nearly all cells in vivo are exposed to a three-dimensional(3-D)environment made up of various types of cells and complex Extracellular Matrix(ECM).To some degree,the functions of organs are determined by the specific 3-D cell arrangement.In traditional two-dimensional(2-D)culture systems,cells can only attach to the flat solid surface of a petri dish,lacking cell-cell and cell-ECM interactions;as such,the formation of 3-D structure cannot be studied,not to mention the interaction of cancer cells with the 3-D environment.Thus,development of an in vitro 3-D model that closely mimics actual environment of tissue has become extraordinarily important for anti-cancer study.In recent years,various 3-D cell culture systems have been explored,but the most popular and effective model is multicellular tumor spheroids.Multicellular tumor spheroids could better mimic the actual 3-D spatial structure organization presented by solid tumors.Several methods have been developed over the years to form multicellular tumor spheroids.Microfluidic encapsulation of cells within hydrogel materials has attracted more attention because it has quite a few advantages over other technologies.In this work,we describe a microfluidic device used as a robust platform for generating core-shell hydrogel microspheres with precisely controlled sizes and varied components of hydrogel matrices.The microfluidic device provides a more efficient platform to manipulate cells and its environment.To gain better understanding of the governing mechanism of microsphere formation,computational models based on multiphase flow were developed to numerically model the droplet generation and velocity field evolution process with COMSOL Multiphysics software.Modeling results show good agreement with experiments in size dependence on flow rate as well as effect of vortex flow on microsphere formation.The details are as follows:(1)Design of microfluidic chip: The pattern of PDMS chip was designed with the L-edit software(Tanner EDA).We use soft lithographic technique and oxygen plasma treatment to fabricate the PDMS microfluidic chip for droplets formation.The dynamics of droplet generation in the microfluidic device was investigated by both experiment and simulation.In this way,we can make sure that droplets can be generated stably.The simulation results can be used as reference parameters for chip experiments.(2)Hydrogel microspheres formation: We have successfully obtained hydrogel microspheres with stable and uniform size,which verify simulation results effectively.In addition,through experiment and numerical simulation,the effect of the flow rate of oil phase on droplet formation was investigated.We are also studying the distribution of the fluorescence intensity over the channel cross-section in the co-flow area at different ratio of two aqueous phases.Therefore,our microfluidic chip can generate hydrogel droplets with variable volume ratios of core to shell and adjust the ratio in real-time.(3)Cell-laden hydrogel microspheres formation:Combined with a controlled low-temperature cooling apparatus,we have successfully obtained cell-laden hydrogel microspheres with core-shell structure.Experiments found that the flow rate of oil 2 has a significant effect on the formation of core-shell structure.Computational models based on multiphase flow were developed to numerically model the velocity field evolution process with COMSOL Multiphysics software.Increasing oil phase velocity results in an unstable vortex flow formation,leading to the destruction of the core-shell interface(i.e.layered structure).Modeling results show good agreement with experiments in effect of vortex flow on microsphere formation.The heterogeneous core-shell microspheres provide well mimicked ECM microenvironment to observe cell-cell and cell-ECM interactions.Moreover,viability of encapsulated cells is evaluated by standard Acridine orange and propidium iodide staining.